Previous work in this laboratory had established that the Mg2 ion -ATPase activity of Acanthamoeba myosin I could be activated by actin only in the presence of another protein factor derived from the amoeba. The active component was called cofactor protein. We have now partially purified the cofactor activity and demonstrated that it is a protein kinase that specifically phosphorylates the heavy chain of Acanthamoeba myosins IA, IB and IC. The phosphorylated myosins have unchanged activities as Ca2 ion minus, (K ion, EDTA)- and Mg2 ion -ATPases but differ from the non-phosphorylated enzyme in being highly activated by F-actin. Although the kinase is still not pure, activity seems to be attributable to a protein of molecular weight about 105,000. The kinase requires Mg2 ion but is unaffected by Ca2 ion or cyclic nucleotides. The kinase has no activity with Acanthamoeba myosin II, platelet myosin or smooth muscle myosin as substrates. It differs from other known myosin kinases that similarly stimulate actin-activated myosin Mg2 ion -ATPase in that the Acanthamoeba kinase specifically phosphorylates the heavy chain while the other myosin kinases specifically phosphorylate 20,000-dalton light chains.